B19—Gly—B20人胰岛素的分离纯化及性质研究

目的：研究胰岛素B链羧端的自由度对于胰岛素与受体相互作用的影响。方法：在人胰岛素B链羧端β转角区回折点B19与B20之间插入了一个Gly。结果：B19-Gly-B20人胰岛素原的表达产物占细胞总蛋白量的30％,B19－Gly-B20人胰岛素的受体活性是标准猪胰岛素的121％。结论;以较高的受体结合活性获得了高纯度的B19－Gly-B20人胰岛素。     

B23-Gly-B24人胰岛素的分离纯化及性质研究

为了研究胰岛素B链羧端的自由度对于胰岛素与受体相互作用的影响。在人胰岛素B链羧端（转角区回折点B23和B24之间插入一个Gly。B23－Gly-B24人胰岛素原在大肠杆菌的表达产物占细胞总蛋白量的28％，B23－Gly-B24人胰岛素的受体活性分别是标准猪胰岛素的122％和人胰岛素的111％。说明在较高的受体结合活性获得了高纯度的B23－Gly-B24人胰岛素。     

B9位丝氨酸残基是胰岛素的葡萄糖代谢作用所必需的(英文)

目的:研究B9和B10位氨基酸残基在胰岛素分子中的重要性。方法:在分离的大鼠脂肪细胞中测定[B9Glu,B10Asp]胰岛素的受体结合活性、葡萄糖吸收活性和脂生成活性。用Western印迹法检测[B9Glu,B10Asp]胰岛素对葡萄糖转运蛋白-4(Glut4)转位和胰岛素受体自磷酸化的刺激活性。结果:[B9Glu,B10Asp]胰岛素的受体结合活性和刺激葡萄糖吸收、脂生成、Glut4转位和胰岛素自磷酸化活性分别是天然胰岛素的31％、45％、40％、58％和46％。结论:B9位丝氨酸残基是胰岛素的葡萄糖代谢作用所必需的。     

瑞格列奈治疗2型糖尿病的临床疗效观察

瑞格列奈是甲基甲胺苯甲酸家族的第一个新型口服抗糖尿病药 ,是非磺脲类促胰岛素分泌剂 ,通过与胰岛B细胞膜上特异性受体结合 ,刺激胰岛素分泌而发挥作用。口服后 ,可被迅速吸收 ,对胰岛素分泌的促进作用较快 ,1h达高峰 ,但持续时间短 ,其半衰期约 1h。可在 2型糖尿病患者中模拟生理性胰岛素分泌 ,有效的控制餐后高血糖。瑞格列奈在肝脏代谢成非活性物质 ,主要通过胆汁排泄。1　对象与方法1 1　对象　根据WHO(1999)标准诊断的 2 0 0 1年 10月～2 0 0 2年 12月我科住院及门诊 2型糖尿病患者 6 9人 ,其中男性 36人 ,女性 33人 ,年龄在 35～ …     

B9位丝氨酸残基是胰岛素的葡萄糖代谢作用民必需的

目的：研究B9和B10位氨基酸残基在胰岛素分子中的重要性。方法：在分离的大鼠脂肪细胞中测定[B9Glu,B10Asp]胰岛素的受体结合活性、葡萄糖吸收活性和脂生成活性。用Western印迹法检测[B9Glu,B10Asp]胰岛素对葡萄糖转运蛋白－4（Glut4)转位和胰岛素受体自磷酸化的刺激活性。结果：[B9Glu,B10Asp]胰岛素的受体结合活性和刺激葡萄糖吸收、脂生成、Glut4转位和胰岛素自磷酸化活性分别是天然胰岛素的31％、45％、40％、58％和46％。结论：B9位丝氨酸残基是胰岛素的葡萄糖代谢作用所必需的。     

小C肽人胰岛素原类似物(B-Arg-Arg-A)和人胰岛素原(B-C-A)A、B链重组条件的研究

目的 :获得更佳的变复性条件和提高人胰岛素原的重组率。方法 :采用特有的重组方法 ,将小C肽人胰岛素原类似物 (B Arg Arg A)与人胰岛素原 (B C A)进行重组条件的比较。 结果 :发现二者在 pH10 .6左右处重组率达到最大 ,且在相同的条件下B Arg Arg A蛋白质的重组率 (94% )较B C A蛋白质 (82 % )的高。结论 :进一步验证了“胰岛素的A、B链含有足够使二硫键正确配对的结构信息”的论点。     

特发性血小板减少性紫癜与人细小病毒B_(19)的关系

目的　探讨特发性血小板减少性紫癜 (ITP)与人细小病毒B19感染性的关系。方法　采用巢式聚合酶链反应技术分别对 6 3例ITP患儿的血清、38例ITP患儿的骨髓液检测人细小病毒B19 DNA。并与对照组进行对比。结果　① 6 3例ITP静脉血中人细小病毒B19 DNA阳性 12例 ,阳性率为 19 1% ,186例健康儿童 11例阳性 ,阳性率为 5 91%。血清的ITP组与对照组差异有显著意义 (χ2 =9 6 8,P <0 0 1)。② 38例ITP患儿的骨髓液中人细小病毒B19 DNA 16例阳性 ,阳性率为 4 2 1% ,对照组 30例阳性 3例 ,阳性率为10 % ,骨髓ITP组与对照组差异有显著意义 (χ2 =8 5 8,P <0 0 1)。③血清的ITP组 12例阳性病例中 ,11例是急性ITP ,阳性率为 2 3 4 % ;1例是慢性ITP ,阳性率为 5 9% ,差异有显著意义 (χ2 =3 0 9,P <0 0 5 )。骨髓的ITP组急性 2 9例 ,阳性 15例 ,阳性率为 5 1 7% ;慢性 9例 ,阳性 1例 ,阳性率为 11 1%。ITP组与对照组差异有显著意义 (χ2 =4 6 5 ,P <0 0 5 )。男女之间无差异。结论　①ITP人群有较高的B19感染率 ,一部分ITP病儿与人细小病毒B19感染密切相关。②临床上一部分ITP患儿的发病可能为人细小病毒B19感染所导致。③B19感染相关性ITP以急性ITP为主。     

诺和锐30

[通用名称]insulin aspart 30 injection，门冬胰岛素30注射液 [化学成分]本品含30％可溶性门冬胰岛素和70％精蛋白门冬胰岛素，其活性成分为门冬胰岛素（利用生物技术将人胰岛素氨基酸链B28位的脯氨酸用天门冬氨酸替代）。1 IU（单位）相当于0．035mg不含无机盐的无水门冬胰岛素。     

DAB389(Gly4Ser)2-αMSH重组蛋白的构建表达及特异性细胞毒性研究*

目的构建重组毒素DAB389(Gly4Ser)2-αMSH,探讨其对过度表达αMSH受体的几种癌细胞的特异杀伤作用.方法利用含有His·Tag和白喉毒素的基因序列作上游引物、含有αMSH全序列和(Gly4Ser)2的互补序列基因作下游引物,以pET28a-DAB389(Gly4Ser)2-EGF为模板,PCR扩增得到DAB389(Gly4Ser)2-αMSH片段,插入原核表达载体pET28a的相应位点,构建重组表达载体并在大肠杆菌中表达重组融合蛋白DAB389(Gly4Ser)2-αMSH.用SDS-PAGE和Western blot鉴定表达的重组蛋白,用结晶紫法检测重组蛋白对几种癌细胞和正常细胞的毒性作用.结果构建了含有His·Tag重组表达质粒pET28a-DAB389(Gly4Ser)2-αMSH;SDS-PAGE表明,重组蛋白相对分子质量为45.32×103,其表达量占菌体蛋白总量的29.2%;Western blot分析显示,重组蛋白能特异地与抗白喉抗体结合;细胞活性检测表明重组蛋白对SP2/0,HeLa,A549,A375这4种癌细胞有明显的杀伤作用,4种癌细胞的IC50值分别是3.138,2.736,7.243和4.672 μg·mL-1.结论成功构建了具有生物学活性的重组蛋白DAB389(Gly4Ser)2-αMSH,为进一步的靶向性药物研究奠定了基础.     

刊授医学继续教育测验题上期(2003-3)答案

一、《β受体阻滞剂在慢性心衰中的应用》 :1 E2 D　 3 A　 4 E　 5 B　 6 D　 7 A　 8 D　 9 D　10 D　 11 E　 12 B　 13 B　 14 D　 15 D　 16 A　17 B　 18 B　 19 A　 2 0 C二、《治疗药物监测与个体化用药》 :1 D 2 C3 B　 4 E　 5 B　 6 D　 7 C　 8 E　 9 D　 10 E　11 D　 12 A　 13 D　 14 C　 15 D　 16 C　 17 E　18 A　 19 C　 2 0 E三、《我国全科医学教育体系概述》 :1 E 2 A3 D　 4 C　 5 D　 6 A　 7 C　 8 B　 9 A　 10 B　11 E　 12 B　 13 A　 14 E　 15 C　 16 E…     

Monomeric destetrapeptide human insulin from a precursor expressed in Saccharomyces cerevisiae

Abstract: Destetrapeptide insulin (DTI, human insulin with B27–30 removed) was obtained from a monomeric precursor (MIP) expressed in Saccharomyces cerevisiae through tryptic transpeptidation in the presence of synthetic tetrapeptide Gly‐Phe‐Phe‐Tyr. The in vivo biological activity of DTI, determined by mouse convulsion assay, is 22 IU/mg. Its binding activity with insulin receptor on human placental membrane is 80% and its in vitro biological activity, determined by free fat cell assay, is 77%. Compared with native insulin, DTI molecules do not associate in solution but exist in the monomeric form, thus leading to its rapid utilization in vivo.     

The role of the C-terminus of the insulin B-chain in modulating structural and functional properties of the hormone

In memoriam Howard S. Tager Within the scope of structure-function studies on the proteohormone insulin, the role of the C-terminal segment B26-B30 for self-association and receptor interaction was analyzed. Insulin derivatives with modifications in the region B26-B30 were synthesized by trypsin-catalyzed coupling reactions of des-(B23-B30)-insulin with synthetic peptides. The peptides were obtained by Fmoc solid-phase peptide synthesis. Insulins with multiple amino acid → glycine substitutions were examined to distinguish between the influence of the side chains and the influence of the main chain in positions B27-B30 on the self-association of the hormone. The analogues [Gly^{B27,B28,B29,B30}]insulin and [Gly^B27,B28,B30]insulin exhibit relative receptor affinities of 80% and self-associate. The successive extension of [Ala^B26]des-(B27-B30)-insulin-B26-amide (relative receptor binding 273%) with amino xids corresponding to the native sequence B27-B30 showed the influence of the length of the B-chain on receptor affinity: the extension by B27-threonine amide reduces receptor binding to 71%, all further prolongations have only small effects on the binding. The effect of the B28-side chain on main-chain conformation, self-association and receptor binding was examined with [X^B28]des-(B29-B30)-insulin-B28-amides (X = Phe, Gly, D-Pro). While the glycine and D-proline analogues (relative binding 104 and 143%, respectively) retain the self-association properties typical of insulin, [Phe^B28]des-(B29-B30)-insulin-B28-amide (relative binding 50%) shows diminished self-association. The backbone-modified insulin derivative [Sar^B26]des-(B27-B30)-insulin-B26-amide (sarcosine =N-methylglycine) exhibits an unexpectedly high receptor affinity of 1100% which demonstrates that the B26-amide hydrogen of the native hormone is not important for receptor binding. © Munksgaard 1995.     

An insulin analogue with γ-amino butyric acid substitution for A13Leu-A14Tyr

Abstract: An insulin A chain analogue, [A13–14 GABA, A21 Ala]A chain, for which the dipeptide Leu-Try at A13-A14 was substituted by a non-coded amino acid, γ-amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid-phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13–14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50%in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C–N peptide bond by an unnatural C–C bond may have general meaning in structure and function studies of other proteins.     

Insulin aspart (AspB28 human insulin) derivatives formed in pharmaceutical solutions

Purpose. To isolate and identify the main insulin aspart (Asp^B28 human insulin) derivatives formed in pharmaceuticals (pH 7.4 at 5°C), to estimate rates of formation, and to determine their biologic potencies. Methods. Insulin aspart derivatives have been isolated by reversed-phase high-performance liquid chromatography (RP-HPLC), and identified by RP-HPLC, peptide mapping, amino acid analysis, mass spectrometry, and N-terminal amino acid sequence analysis. Results. The main derivatives formed were isoAsp^B28, isoAsp^B3, Asp^B3, and desPhe^B1-N-oxalyl-Val^B2 insulin aspart. At 5°C, the rate constants were 0.00028/month for isoAsp^B28 and isoAsp^B3, 0.00024/month for Asp^B3, and 0.00013/month for desPhe^B1-N-oxalyl-Val^B2 derivatives of insulin aspart. Unexpectedly, the rate of isomerization of B28 was high compared to the rate of B3 deamidation at both 5°C and 45°C. The N-terminal and especially the C-terminal of the B-chain are highly flexible, which may explain the high rate of isoAsp^B28 formation and that deamidation of Asn^B3 occurs. All the derivatives had full in vivo biologic potencies. Conclusion. Except for isoAsp^B28 insulin aspart, the main derivatives formed in pharmaceuticals of insulin aspart and human insulin at pH 7.4 are similar. They are all fully active in vivo. In proteins, flexibility of the polypeptide chain seems more important than sequence in the formation of succinimides.     

The Structural and Functional Role of the B‐chain C‐terminal Arginine in the Relaxin‐3 Peptide Antagonist,R3(BΔ23‐27)R/I5

Relaxin‐3, a member of the insulin superfamily, is involved in regulating stress and feeding behavior. It is highly expressed in the brain and is the endogenous ligand for the receptor RXFP3. As relaxin‐3 also interacts with the relaxin receptor RXFP1, selective agonists and antagonists are crucial for studying the physiological function(s) of the relaxin‐3/RXFP3 pair. The analog R3(BΔ23‐27)R/I5, in which a C‐terminally truncated human relaxin‐3 (H3) B‐chain is combined with the INSL5 A‐chain, is a potent selective RXFP3 antagonist and has an Arg residue remaining on the B‐chain C‐terminus as a consequence of the recombinant protein production process. To investigate the role of this residue in the RXFP3 receptor binding and activation, the analogs R3(BΔ23‐27)R/I5 and R3(BΔ23‐27)R containing the B‐chain C‐terminal Arg as well as R3(BΔ23‐27)/I5 and R3(BΔ23‐27), both lacking the Arg, were chemically assembled and their secondary structure and receptor activity assessed. The peptides generally had a similar conformation but those with the extra Arg residue displayed a significantly increased affinity for the RXFP3. Interestingly, in contrast to R3(BΔ23‐27)R and R3(BΔ23‐27)R/I5, the peptide R3(BΔ23‐27) is a weak agonist. This suggests that the C‐terminal Arg, although increasing the affinity, alters the manner in which the peptide binds to the receptor and thereby prevents activation, giving R3(BΔ23‐27)R/I5 its potent antagonistic activity.     

Implications of replacing peptide bonds in the COOH-terminal B chain domain of insulin by the Ψ(CH2-NH) linker

To evaluate more thoroughly the importance of main-chain structure and flexibility in ligand interactions with the insulin receptor, we undertook to synthesize analogues with reduced peptide bonds in the COOH-terminal B chain domain of the hormone (a stable, but adjustable β-strand region). By use of solid-phase, solution-phase and semisynthetic methods, analogues were prepared in which Arg^B22 of des-octapeptide(B23–B30)-insulin was extended by the sequences Gly-Phe-(CH₂-NH)-Phe-NH₂, Gly-Gly-(CH₂-NH)-Phe-Phe-NH₂, Gly-Phe-(CH₂-NH)-Phe-Phe-Thr-Pro-Ala-Thr-OH, and Gly-Phe-Phe-(CH₂-NH)-Phe-Thr-Pro-Ala-Thr-OH, and were studied with respect to their abilities both to interact with the hepatocyte insulin receptor and to form soluble anion-stabilized hexamers in the presence of Co²⁺ and phenol. Additional analogues of des-pentapeptide(B26–B30)-insulin were also examined. Overall, our results show that, whereas all analogues retain considerable ability to form organized metal ion-coordinated complexes in solution, the reduction of peptide bonds both proximal and distal to the critical side chain of Phe^B25 results in analogues with severely diminished receptor binding potency. We conclude that the peptide carbonyls from both Phe²⁴ and Phe^B25 are important for insulin–receptor interactions and that the structural organization of the region when insulin is bound to its receptor differs from that occurring during simple monomer–monomer and higher-order interactions of the hormone.     

Structure-based design of mimetics for granulocyte-macrophage colony stimulating factor (GM-CSF)

Granulocyte-macrophage colony stimulating factor (GM-CSF) activity has been linked to pro-inflammatory effects in autoimmune syndromes, such as rheumatoid arthritis. Thus GM-CSF mimetics with antagonist activity might play a therapeutic role in these diseases. The human GM-CSF core structure consists of a four alpha-helix bundle, and GM-CSF activity is controlled by its binding to a two-subunit receptor. A number of residues located on the B and C helices of GM-CSF are postulated to interact with the alpha chain of the GM-CSF receptor (GM-CSFR). Several approaches have been successfully utilized to develop peptide mimetics of this site, including peptides from the native sequence, a peptide derived from a recombinant antibody (rAb) light chain which mimicked GM-CSF receptor binding activity, and structurally guided de novo design. Analysis of the rAb light chain had suggested mimicry of GM-CSF with residues mostly contributed by the CDR I region. Key residues involved in CDR I peptide/GM-CSFR binding were identified by truncation and alteration of individual residues, while the structural elements required to antagonize the biological action of GM-CSF were separately tested in binding and inhibitory activity assays of multiple cyclic analogues. A peptide designed to retain the loop conformation of the CDR I region of the rAb light chain competed with GM-CSF for both antibody and receptor binding, but the role of specific residues in antibody versus receptor binding differed markedly. These studies suggest that structural analysis of peptide mimetics can reveal differences in receptor and antibody binding, perhaps including key interactions that impact binding kinetics. Peptide mimetics of other four-helix bundle cytokines are reviewed, including helical and reverse turn mimetics of helical structures. Use of peptide mimetics coupled with structural and kinetic analysis provides a powerful approach to identifying important receptor-ligand interactions, which implications for rational design of novel therapeutics.     

Metformin (Glucophage) inhibits tyrosine phosphatase activity to stimulate the insulin receptor tyrosine kinase

Metformin is a commonly used anti-diabetic but whether its mechanism involves action on the insulin receptor or on downstream events is still controversial. With a time course that was slow compared with insulin action, metformin increased tyrosine phosphorylation of the regulatory domain of the insulin receptor (specifically, tyrosine residues 1150 and 1151). In a direct action, therapeutic levels of metformin stimulated the tyrosine kinase activity of the soluble intracellular portion of the beta subunit of the human insulin receptor toward a substrate derived from the insulin receptor regulatory domain. However, metformin did not alter the order of substrate phosphorylation by the insulin receptor kinase. Using a Xenopus oocyte preparation, we simultaneously recorded tyrosine kinase and phosphatase activities that regulate the insulin receptor by measuring the tyrosine phosphorylation and dephosphorylation of peptides derived from the regulatory domain of the human insulin receptor. In an indirect stimulation of the insulin receptor, metformin inhibited endogenous tyrosine phosphatases and purified human protein tyrosine phosphatase 1B that dephosphorylate and inhibit the insulin receptor kinase. Thus, there was evidence that metformin acted directly upon the insulin receptor and indirectly through inhibition of tyrosine phosphatases.     

Quantitative dissociation of glucose transport stimulation and insulin receptor tyrosine kinase activation in isolated adipocytes with a covalent insulin dimer (B29,B29'-suberoyl-insulin)

The covalent insulin dimer B29,B29'-suberoyl-insulin was investigated for its effects on insulin receptor binding, insulin receptor tyrosine kinase activity and glucose transport in isolated adipose cells. The dimer stimulated glucose transport (initial 3-O-methylglucose uptake rate) to the same extent as insulin did (basal rate, 35 +/- 3 pmol/sec/microliter lipid; insulin, 380 +/- 27; B29,B29'-suberoyl-insulin, 369 +/- 24, means +/- S.E.), although at higher concentrations (EC50 1.94 +/- 0.64 nM versus 0.1 +/- 0.02 with insulin). In contrast, the dimer only partially (23%) mimicked insulin's effect on phosphate incorporation into insulin receptors immunoprecipitated after equilibration of cells with [32P]phosphate. Similarly, insulin receptor tyrosine kinase as assessed by receptor autophosphorylation and phosphorylation of the substrate poly-(Glu/Tyr) was not fully activated by treatment of cells with the insulin dimer (31 and 42% of the effect of insulin, respectively) in concentrations which maximally activate glucose transport and give rise to full insulin receptor occupancy (5 X 10(-7) M). Further, the dimer activated the receptor tyrosine kinase in solubilized purified insulin receptor preparations from adipose cells to only 25% of the effect of insulin (EC50 32.0 +/- 16 versus 1.9 +/- 1.0 nM with insulin) in spite of full receptor occupancy. Binding of the dimer to insulin receptors followed single site binding kinetics, indicating that the derivative is unable to induce negative cooperativity of the insulin receptor. It is concluded that a partial phosphorylation of insulin receptors and a submaximal tyrosine kinase activation are sufficient for full stimulation of glucose transport in the adipocyte. Further, it is suggested that negative cooperativity of the insulin receptor and activation of its tyrosine kinase require a similar conformational change of the receptor protein.     

Discovery of the first non-peptide full agonists for the human bradykinin B(2) receptor incorporating 4-(2-picolyloxy)quinoline and 1-(2-picolyl)benzimidazole frameworks

In the course of our studies on non-peptide bradykinin (BK) B(2) receptor ligands, it was suggested that the 4-substituent of the quinoline ring may play a critical role in determining binding affinities for human and guinea pig B(2) receptors, as well as agonist/antagonist properties. We carried out an extensive investigation to elucidate the structure-activity relationships (SAR) for this key pharmacophore. Introduction of lower alkoxy groups to the 4-position of the quinoline ring of 3 led to the identification of 4-ethoxy derivative 22b as a unique partial agonist. This compound significantly stimulated inositol phosphates (IPs) formation in Chinese hamster ovary cells expressing the cloned human B(2) receptor at concentrations greater than 10 nM and displayed one-tenth of the intrinsic activity of BK. The agonist activity of 22b was selective for the B(2) receptor and was inhibited by selective peptide and non-peptide B(2) antagonists. On the other hand, 22b strongly suppressed BK-induced IPs formation through the cloned human B(2) receptor. Further studies on the key pharmacophore led to identification of a 2-picolyloxy moiety as a powerful agonist switch, leading to the discovery of a potent and efficacious non-peptide B(2) agonist, 19a. Successive optimization of the acyl side chain afforded 38, which exhibited full agonist activity on stimulation of IPs formation. Furthermore, this strategy could be applied successfully to the benzimidazole series. The representative 1-(2-picolyl)benzimidazole derivative 47c increased PGE(2) production at a 1 microM concentration to the same level as the maximum effect of BK. Thus, we have established the medicinal chemistry modifications required to convert our highly potent non-peptide B(2) antagonists to agonists with potent efficacy.